Synthesis of 2-alkylcysteine via substituted thiazoline amide

ABSTRACT

Non-natural amino acids such as 2-alkylated amino acids allow for the synthesis of a wider variety of peptidal and non-peptidal pharmaceutically active agents. In one embodiment, the present invention relates to a method of preparing 2-alkylcysteine comprising condensing cysteine with an aryl nitrile to form a 2-arylthiazoline-4-carboxylic acid, forming a 2-arylthiazoline-4-amide using an amine group comprising at least one substituted or unsubstituted alkyl group that comprises one or more chiral carbon atoms, and alkylating at the 4-position of the thiazoline ring to form a 2-aryl-4-alkyl-thiazoline-4-amide. The present invention also discloses a method of preparing a class of iron chelating agents related to desferrithiocin, all of which contain a thiazoline ring. In one embodiment, an aryl nitrile or imidate is condensed with cysteine or a 2-alkyl cysteine.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.60/381,012, 60/381,021, 60/380,894, 60/380,910, 60/380,880, 60/381,017,60/380,895, 60/380,903, 60/381,013, 60/380,878 and 60/380,909, all ofwhich were filed May 15, 2002. This application also claims the benefitof U.S. Provisional Application No. 60/392,833, filed Jun. 27, 2002. Theentire teachings of the above-referenced applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Alpha-amino acids are useful starting materials in the synthesis ofpeptides, as well as non-peptidal, pharmaceutically activepeptidomimetic agents. In order to enable the synthesis of a largenumber of compounds from an amino acid precursor, it is advantageous tohave naturally occurring and non-naturally occurring amino acids.Non-naturally occurring amino acids typically differ from natural aminoacids by their stereochemistry (e.g., enantiomers), by the addition ofalkyl groups or other functionalities, or both. At this time, theenantiomers of naturally occurring amino acids are much more expensivethan the naturally occurring amino acids. In addition, there are only alimited number of commercially available amino acids that arefunctionalized or alkylated at the alpha-carbon, and often synthesesinvolve the use of pyrophoric or otherwise hazardous reagents. Moreover,the syntheses are often difficult to scale up to a commercially usefulquantity. Consequently, there is a need for new methodologies ofproducing such non-naturally occurring amino acids.

Non-naturally occurring amino acids of interest include the (R)- and(S)-isomers of 2-methylcysteine, which are used in the design ofpharmaceutically active moieties. Several natural products derived fromthese isomers have been discovered in the past few years. These naturalproducts include desferrithiocin, from Streptomyces antibioticus; aswell as tantazole A, mirabazole C, and thiangazole, all from blue-greenalgae. These compounds have diverse biological activities ranging fromiron chelation to murine solid tumor-selective cytotoxicity toinhibition of HIV-1 infection.

Desferrithiocin, deferiprone, and related compounds represent an advancein iron chelation therapy for subjects suffering from iron overloaddiseases. Present therapeutic agents such as desferroxamine requireparenteral administration and have a very short half-life in the body,so that patient compliance and treatment cost are serious problems forsubjects receiving long-term chelation therapy. Desferrithiocin andrelated compounds are effective when orally administered, therebyreducing patient compliance issues. Unfortunately, (S)-2-methylcysteine,which is a precursor to the more active forms of desferrithiocin andrelated compounds, remains a synthetic challenge. Therefore, there is aneed for novel methods of producing 2-methylcysteine at a reasonablecost, and means of isolating the desired enantiomer.

SUMMARY OF THE INVENTION

A useful and efficient method of preparing a 2-alkylcysteine involvescondensing cysteine with an aryl nitrile to form a2-arylthiazoline-4-carboxylic acid, forming a2-arylthiazoline-4-carboxamide using an amine group comprising at leastone substituted or unsubstituted alkyl group that comprises one or morechiral carbon atoms, and alkylating at the 4-position of the thiazolinering to form a 2-aryl-4-alkyl-thiazoline-4-carboxamide. The thiazolineamide has chiral templates, which can provide face selectivity andconsequently desired stereochemistry, during the delivery of an alkylgroup to the 4-position of the thiazoline ring. The chiral templatepresent in the thiazoline amide preferably produces an enantiomericexcess of one isomer.

In one embodiment, the present invention relates to a method ofpreparing a 2-alkylated cysteine represented by Structural Formula (I):

or a salt thereof, wherein R₁ is a substituted or unsubstituted alkylgroup, the method comprising:

(a) coupling a compound (which may be an (R) or (S)-isomer or a mixturethereof) represented by Structural Formula (II):

 with a substituted or unsubstituted aryl nitrile of the formula Ar—CN,wherein Ar is a substituted or unsubstituted aryl group; thereby forminga substituted thiazoline carboxylic acid represented by StructuralFormula (III):

(b) reacting the substituted thiazoline carboxylic acid with an aminerepresented by Structual Formula (IV):

 wherein R^(*) is a substituted or unsubstituted alkyl group comprisingone or more chiral carbon atoms and R₂ is a substituted or unsubstitutedalkyl or aryl group (optionally with one or more chiral carbons);thereby forming a substituted thiazoline amide represented by StructuralFormula (V):

(c) alkylating the substituted thiazoline amide with one or more basesand R₁X, wherein X is a leaving group and R₁ is as defined above;thereby forming an alkylated substituted thiazoline amide represented byStructural Formula (VI):

(d) hydrolyzing the alkylated substituted thiazoline amide, therebyforming an alkylated substituted thiazoline carboxylic acid or a saltthereof, the anion of which is represented by Structural Formula (VII):

(e) reacting the alkylated substituted thiazoline carboxylic acid withacid (preferably an inorganic acid such as HCl, HBr or sulfuric acid),thereby forming the 2-alkylated cysteine represented by StructuralFormula (I).

The methods described above may additionally comprise the step ofpurifying or ultrapurifying the alkylated substituted thiazolinecarboxylic acid or the alkylated substituted thiazoline amide. Purifyingor ultrapurifying the acid or ester can comprise further resolving theenantiomers or diasteromers of the alkylated substituted thiazolinecarboxylic acid or the alkylated substituted thiazoline amide.Alternatively, the 2-alkylated cysteine itself can be resolved.Additionally, the methods can comprise the isolation of the enantiomersof the synthesis products. Preferably, the (S)-enantiomer of2-alkylcysteine is isolated, for example, (S)-2-methylcysteine.

In another aspect, the present invention relates to a method ofpreparing a compound represented by Structural Formula (VIII):

or a salt thereof, the method comprising:

(a) coupling a compound (which may be an (R) or (S)-isomer or a mixturethereof) represented by Structural Formula (IX):

 with a substituted of unsubstituted aryl nitrile of the formula Ar—CN,wherein Ar is a substituted or unsubstituted aryl group; thereby forminga substituted thiazoline carboxylic acid represented by StructuralFormula (X):

(b) reacting the substituted thiazoline carboxylic acid with an aminerepresented by Structual Formula (XI):

 wherein R^(*) is a substituted or unsubstituted alkyl group comprisingone or more chiral carbon atoms and R₂ is a substituted or unsubstitutedalkyl or aryl group; thereby forming a substituted thiazoline amiderepresented by Structural Formula (XII):

(c) alkylating the substituted thiazoline amide with one or more basesand CH₃X, wherein X is a leaving group; thereby forming an alkylatedsubstituted thiazoline amide represented by Structural Formula (XIII):

(d) hydrolyzing the alkylated substituted thiazoline amide, therebyforming an alkylated substituted thiazoline carboxylic acid or a saltthereof, the anion of which is represented by Structural Formula (XIV):

(e) optionally, purifying the (S)-isomer of the alkylated substitutedthiazoline carboxylic acid;

(f) reacting the (S)-isomer of the alkylated substituted thiazolinecarboxylic acid with acid, thereby forming (S)-2-methylcysteine; and

(g) coupling (S)-2-methylcysteine with 2,4-dihydroxybenzonitrile,thereby forming the compound represented by Structural Formula (VIII).

Advantages of the present invention include the facile synthesis of a2-alkyl cysteine from cysteine, an inexpensive and readily availablestarting material. 2-Methylcysteine prepared by the method of thepresent invention can be coupled to 2,4-dihydroxybenzonitrile to form4′-hydroxydesazadesferrithiocin, also referred to as4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid, an iron chelating agent.

DETAILED DESCRIPTION OF THE INVENTION

A useful and efficient method of preparing 2-alkylcysteine involvescondensing cysteine with an aryl nitrile to form a2-arylthiazoline-4-carboxylic acid, forming a 2-arylthiazoline-4-amideusing an amine group comprising at least one substituted orunsubstituted alkyl group that comprises one or more chiral carbonatoms, and alkylating at the 4-position of the thiazoline ring to form a2-aryl-4-alkyl-thiazoline-4-amide (i.e., an alkylated substitutedthiazoline amide). The resulting enantiomers of the product can befurther purified and isolated into pure or substantially pure enantiomercomponents by a number of methods.

The condensation of an aryl nitrile and cysteine typically occurs in apolar, protic solvent in the presence of an excess of base. Typically,the aryl nitrile and cysteine are refluxed together for several hours,such as 1-20 hours, 2-15 hours, 4-10 hours, or 6-8 hours. Refluxingpreferably occurs in an inert atmosphere, such as nitrogen or argon.Preferred aryl nitrites include aryl nitrites where the aryl group is asubstituted or unsubstituted phenyl group. Phenyl and heteroaryls, suchas pyridines and thiazolines, are preferred aryl groups. Suitable polar,protic solvents include, but are not limited to, water, methanol,ethanol, formic acid, acetic acid, dimethylformamide, N-ethylacetamide,formaldehyde diethyl acetal, and long chain alcohols (e.g., propanol andisopropanol). An alcohol, such as methanol or ethanol, is a preferredsolvent. Suitable bases include secondary and tertiary amines such asdimethylamine, diethylamine, trimethylamine, triethylamine,diisopropylamine, and diisopropylethylamine. The base can be added inexcess, such as one or more equivalents relative to the amount ofcysteine. Suitable amounts of base have at least about one equivalent ofbase, and range from about 1 to about 10, about 1 to about 5, about 1 toabout 3, and about 1 to about 2 equivalents, relative to the amount ofcysteine. In one example, cysteine, benzonitrile, and 5 equivalents oftriethylamine are refluxed in ethanol for about 6-8 hours to obtain a2-phenylthiazoline-4-carboxylic acid.

Alternatively, an aryl imidate (e.g., a benzimidate, where the benzenering can have one or more substituents, as described below) can becondensed with cysteine to form a substituted thiazoline carboxylicacid. The substituted thiazoline carboxylic acid can be formed bycoupling an aryl imidate, such as benzimidate, with a cysteine, such asthe cysteine represented by Structural Formula (II). Typically, couplingof a cysteine or a 2-alkylcysteine with an aryl imidate includesreacting a cysteine (or a related compound) with the aryl imidate underbasic conditions. Acceptable bases include trimethylamine;triethylamine; dimethylamine; diethylamine; diphenylamine;diisopropylamine; diisopropylethylamine; 1,4-diazabicyclo[2.2.2]octane(DABCO); 1,5-diazabicyclo-[4.3.0]-non-5-ene (DBN); and the like.

Aryl imidates can be prepared, for example, for aryl nitriles, arylcarboxylic acids, and aryl amides. Methods of forming aryl imidates arediscussed in U.S. patent application Ser. No. 60/380,909, filed May 15,2002, the entire contents of which are incorporated herein by reference.In one example, an aryl carboxylic acid (e.g., benzoic acid) isconverted into an acid chloride, then an amide, followed by reactionwith a trialkyloxonium hexafluorophosphate or a trialkyloxoniumtetrafluoroborate to form the aryl imidate. In a second example, an arylnitrile is converted into an aryl imidate through reaction with analcohol in the presence of an acid, as is described below.

The thiazoline amide can be produced, for example, by reacting an aminewith a carboxylic acid or with an activated acid (e.g., an ester,anhydride, or acid halide). In one embodiment of the present invention,a substituted thiazoline amide represented by Structual Formula (V) isformed through the reaction of a substituted thiazoline carboxylic acidwith an amine represented by Structual Formula (IV). The aminerepresented by Structual Formula (IV) contains at least one substitutedor unsubstituted alkyl group comprising one or more chiral carbon atoms.Preferred amines include phenylethylamines and heteroaryl chiral aminessuch as, for example, cinchonidine and quinidine. In a preferredembodiment, the amine used in this reaction is substantially opticallypure.

There are several specific pathways by which the substituted thiazolineamide can be formed. In one pathway, the substituted thiazoline amide isformed by reacting the amine with the substituted thiazoline carboxylicacid with at least stoichiometric quantities of a promoting agent (i.e.,with at least one equivalent relative to the substituted thiazolinecarboxylic acid). Preferably, the substituted thiazoline amide is formedby reacting the amine with the substituted thiazoline carboxylic acidwith about one equivalent of a promoting agent. The use of a promotingagent allows the formation of the substituted thiazoline amide attemperatures at or near room temperature. Examples of promoting agentsinclude, but are not limited to, dicyclohexylcarbodiimide (DCC);N,N′-carbonyldiimidazole; POCl₃; TiCl₄; sulfuryl chloride fluoride;benzotriazol-1-yl diethyl phosphate; Ti(OBu)₄; molecular sieves;N,N,N′,N′-tetramethyl(succinimido)uranium tetrafluoroborate;1,1′-carbonylbis(3-methylimidazolium) triflate;2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide(Lawesson's reagent); chlorosulfonyl isocyanate; P₂I₄; pyridiniumsalts-Bu₃N; Bu₃P/PhCNO; SOCl₂; imidazoles; and N-hydroxybenzothiazole(HOBt). Without being held to any particular theory, it is believed thatthe promoting agent DCC acts as a dehydrating agent on the substitutedthiazoline carboxylic acid and encourages the formation of carboxylicacid anhydrides (i.e., (RCO)₂O, where R is the substituted thiazolinegroup), which then react with the amine to form the substitutedthiazoline amide. In a preferred embodiment, the substituted thiazolineamide is formed by the reaction of a substituted thiazoline carboxylicacid with an amine, along with about one equivalent (relative to thesubstituted thiazoline carboxylic acid) of dicyclohexylcarbodiimide(DCC).

In another method, a substituted thiazoline amide is produced bypyrolysis of a mixture of the amine and the substituted thiazolinecarboxylic acid. Carboxylic acids can also be converted to amides byheating with amides of carboxylic acids, sulfonic acids, or phosphoricacids. In one example, a substituted thiazoline amide is formed from thereaction of a substituted thiazoline carboxylic acid with an amide ofcarboxylic acid, sulfonic acid, or phosphoric acid.

In another method, a substituted thiazoline amide is formed from an acylhalide. For example, an acyl halide is formed from the substitutedthiazoline carboxylic acid by reaction with a halogenating agent.Halogenating agents include, but are not limited to, thionyl chloride,PCl₃, PCl₅, PBr₃ and PBr₅. In a preferred embodiment the halogenatingagent is thionyl chloride. The acyl halide is then reacted with anamine, such as the amine represented by Structual Formula (IV),optionally in the presence of a weak base such as pyridine, to form asubstituted thiazoline amide.

The 2-arylthiazoline-4-carboxamide can be alkylated in the presence ofone or more bases, an alkylating agent, and optionally a phase transfercatalyst. Typically, the 2-arylthiazoline-4-amide is reacted with one ormore equivalents (e.g., about 1 to 10, about 1 to 5, about 1 to 3, orabout 1.5 to 2.5 equivalents) of base and one or more equivalents (e.g.,about 1 to 5, about 1 to 2, about 1 to 1.5, or about 1 to 1.1equivalents) of an alkylating agent in a polar, aprotic solvent at about−80 to 40° C., about −50 to 25° C., about −20 to 10° C., or about −5 to5° C.

Alkylating agents represented by the formula R₁X, where R₁ and X are asdefined above. Preferred R₁ groups include substituted or unsubstitutedC1-C4 alkyl groups, for example, methyl or benzyl. The leaving group Xis typically a weak base. Suitable leaving groups include halogen,tosyl, and mesyl, triflyl, brosyl, p-nitrophenyl, and 2,4-dinitrophenylgroups. Halogens include bromine, chlorine, and iodine. Iodine is apreferred leaving group. Preferred bases include alkali metal alkoxides,potassium t-butoxide, sodium methoxide, sodium ethoxide, and sodiumamide. Suitable polar, aprotic solvents include, but are not limited to,dimethylformamide, dimethyl sulfoxide, acetonitrile, acetone,tetrahydrofuran (THF), and hexamethylphosphoramide. Tetrahydrofuran(THF) is a preferred solvent.

In one example, a 2-arylthiazoline-4-carboxamide is reacted with about 2equivalents of base and about 1 equivalent of methyl iodide intetrahydrofuran (THF) at 0° C. to form a2-aryl-4-methyl-thiazoline-4-carboxamide.

Alternatively, the 2-arylthiazoline-4-carboxamide can be alkylated inthe presence of a phase transfer catalyst. Examples of phase transfercatalysts include benzyl triethyl ammonium chloride, benzyl trimethylammonium chloride, benzyl tributyl ammonium chloride, tetrabutylammonium bromide, tetraethyl ammonium bromide, tetrabutyl ammoniumhydrogen sulfate, tetramethyl ammonium iodide, tetramethyl ammoniumchloride, triethylbutyl ammonium bromide, tributyl ethyl ammoniumbromide, tributyl methyl ammonium chloride, 2-chloroethylamine chlorideHCl, bis(2-chloroethyl)amine HCl, 2-dimethylaminoethyl chloride HCl,2-ethylaminoethyl chloride HCl, 3-dimethylaminopropyl chloride HCl,methylamine HCl, dimethylamine HCl, trimethylamine HCl, monoethylamineHCl, diethylamine HCl, triethylamine HCl, ethanolamine HCl,diethanolamine HCl, triethanolamine HCl, cyclohexylamine HCl,dicyclohexylamine HCl, cyclohexylamine HCl, diisopropylethylamine HCl,ethylenediamine HCl, aniline HCl, methyl salicylate, ethyl salicylate,butyl salicylate amyl salicylate, isoamyl salicylate, 2-ethylsalicylate,and benzyl salicylate.

In one embodiment of the present invention, the2-aryl-4-alkyl-thiazoline-4-carboxamide is hydrolyzed to form a2-aryl-4-alkyl-thiazoline-4-carboxylic acid. Substituted amides (e.g.,N- and N,N- substituted amides) can be hydrolyzed with either basic oracid catalysis to form a free carboxylic acid and an amine. For example,2-aryl-4-alkyl-thiazoline-4-carboxamide can be hydrolyzed with water,heat, and either basic or acid catalyst to form2-aryl-4-alkyl-thiazoline-4-carboxylic acid. An example of suitableconditions for hydrolysis is heating the amide under reflux in aqueoushydrochloric acid.

Preferably, 2-aryl-4-methyl-thiazoline-4-carboxylic acid is subsequentlyreacted with acid to form 2-methylcysteine.

The products, either enantiomers or diastereomers, of the above notedsyntheses can be further purified or ultrapurified. In one embodiment,the 2-aryl-4-alkyl-thiazoline-4-carboxamide is further resolved into (R)and (S)-isomers based on the thiazoline 4-carbon. For example, the2-aryl-4-alkyl-thiazoline-4-amide can be purified using the technique ofemulsion crystallization. In one form, the method includes resolving the2-aryl-4-alkyl-thiazoline-4-amide into its (R) and (S)-isomers based onthe thiazoline 4-carbon, isolating the (S)-isomer, hydrolyzing the(S)-isomer to form 2-aryl-4-alkyl-thiazoline-(S)-4-carboxylic acid, andreacting the 2-aryl-4-alkyl-thiazoline-(S)-4-carboxylic acid with acidto form a (S)-2-alkylcysteine. Alternatively, the2-aryl-4-alkyl-thiazoline-4-amide can be hydrolyzed to form a2-aryl-4-alkyl-thiazoline-4-carboxylic acid and then the2-aryl-4-alkyl-thiazoline-4-carboxylic acid can be further resolved intoits (R) and (S)-isomers. For example, the2-aryl-4-alkyl-thiazoline-4-carboxylic acid can be further resolvedusing the technique of emulsion crystallization or by the formation of adiastereomeric salt. Then 2-aryl-4-alkyl-thiazoline-(S)-4-carboxylicacid can be isolated and reacted with acid to form (S)-2-alkylcysteine.Also the 2-aryl-4-alkyl-thiazoline-4-carboxamide can be reacted withacid to form 2-alkylcysteine, the 2-alkylcysteine is then furtherresolved into its (R) and (S)-isomers, and the (S)-isomer is isolated,producing (S)-2-alkylcysteine.

Chiral carboxylic acids can purified through resolution of enantiomersby forming a diastereomeric salt with the chiral carboxylic acid and achiral amine. Suitable chiral amines include arylalkylamines such as1-alkyl-1-aminoalkanes and 1-aryl-1-aminoalkanes. Examples include(R)-1-phenylethylamine, (S)-1-phenylethylamine, (R)-1-tolylethylamine,(S)-1-tolylethylamine, (R)-1-phenylpropylamine, (S)-1-propylamine,(R)-1-tolylpropylamine, and (S)-1-tolylpropylamine. Preferably,(R)-1-phenylethylamine is used to resolve the chiral carboxylic acidmixture. Resolution of chiral compounds using diastereomeric salts isfurther described in CRC Handbook of Optical Resolutions viaDiastereomeric Salt Formation by David Kozma (CRC Press, 2001),incorporated herein by reference in its entirety.

Alternatively, chiral carboxylic acids or chiral amides can be purifiedor ultrapurified by emulsion crystallization, as described in U.S. Pat.Nos. 5,872,259, 6,383,233 and 6,428,583, issued to Reuter, the teachingsof which are incorporated herein in their entirety by reference.Briefly, emulsion crystallization is a process for separating a desiredsubstance from an aggregate mixture. The process involves forming athree phase system, the first phase comprising the aggregate mixture,the second phase being liquid and comprising a transport phase, and thethird phase comprising a surface upon which the desired substance cancrystallize. A chemical potential exists for crystal growth of thedesired substance in the third phase of the system, thereby creating aflow of the desired substance from the first phase through the secondphase to the third phase, where the desired substance crystallizes andwhereby an equilibrium of the activities of the remaining substances inthe aggregate mixture is maintained between the first phase and thesecond phase.

In one example of emulsion crystallization, a solution of the racemicmixture is supersaturated (by either cooling, adding a solvent in whichone or more components are sparingly soluble or by evaporation of thesolution). Ultrasonication typically aids the process of forming anemulsion. The mixture is then seeded with crystals of the desired,optically active acid along with an additional quantity of surfactantand an anti-foaming agent. The desired product usually crystallizes outand can be separated by filtration.

Once the chiral carboxylic acids or chiral amides have been purified,the desired isomer can be isolated. Typically, the (S)-isomer isisolated. For example, (S)-2-methylcysteine, a2-aryl-4-alkyl-thiazoline-(S)-4-carboxylic acid, or a2-aryl-4-alkyl-thiazoline-(S)-4-carboxamide is isolated. Preferably, the2-aryl-4-alkyl-thiazoline-(S)-4-carboxylic acid or the2-aryl-4-alkyl-thiazoline-(S)-4-carboxamide is isolated.

In a preferred embodiment, (S)-2-methylcysteine is formed and isolated.Cysteine or a 2-alkylcysteine such as (S)-2-methylcysteine can becoupled to a substituted or unsubstituted aryl nitrile such as asubstituted or unsubstituted benzonitrile. Preferably, the substituentson benzonitrile will not interfere with the coupling reaction. In oneaspect of the invention, (S)-2-methylcysteine is coupled to2,4-dihydroxybenzonitrile to form4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid (also known as 4′-hydroxydesazadesferrithiocin). In yet anotherembodiment, (S)-2-methylcysteine is coupled to 2-hydroxybenzonitrile toform 4,5-dihydro-2-(2-hydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid (also known as desazadesferrithiocin).

Typically, coupling of cysteine or a 2-alkylcysteine and a substitutedor unsubstituted benzonitrile includes converting the benzonitrile intoa benzimidate. The benzimidate can be formed, for example, by reactingthe benzonitrile with an alcohol such as methanol, ethanol, n-propanol,or isopropanol in the presence of an acid such as hydrochloric acid. Thebenzimidate is then reacted with the cysteine (or related compound)under basic conditions. Acceptable bases include trimethylamine,triethylamine, triphenylamine, and the like. The reaction between thebenzimidate and the cysteine results in the thiazoline (or4,5-dihydrothiazole) containing product. When forming the benzimidatefrom a hydroxylated benzonitrile (e.g., 2,4-dihydroxybenzonitrile), thehydroxyl groups are advantageously protected (e.g., with a substitutedor unsubstituted alkyl or arylalkyl group such as a benzyl group). Theprotecting groups are subsequently cleaved, typically by catalytichydrogenation.

Suitable benzonitriles and benzimidates for use in the above couplingreaction can be synthesized by methods described in U.S. patentapplication Ser. Nos. 60/381,013, 60/380,878 and 60/380,909, filed May15, 2002. The entire contents of these applications are incorporatedherein by reference.

The methods of the claimed invention can be used to manufacture otherrelated desferrithiocin analogs and derivatives. Examples of suchanalogs include those described in U.S. Pat. Nos. 5,840,739, 6,083,966,6,159,983, 6,521,652 and 6,525,080, all issued to Bergeron, the contentsof which are incorporated herein by reference. Additional examples canbe found in International Application Nos. PCT/US93/10936, published asWO 94/1137 on May 5, 1994; PCT/US97/04666, published as WO 97/36885 onOct. 9, 1997; and PCT/US99/19691, published as WO 00/12493 on Mar. 9,2000, the entire contents of which are incorporated herein by reference.

An alkyl group is a hydrocarbon in a molecule that is bonded to oneother group in the molecule through a single covalent bond from one ofits carbon atoms. Alkyl groups can be cyclic, branched or unbranched,and/or saturated or unsaturated. Typically, an alkyl group has one toabout 24 carbons atoms, or one to about 12 carbon atoms. Lower alkylgroups have one to four carbon atoms and include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl.

Aromatic (or aryl) groups include carbocyclic aromatic groups such asphenyl, p-tolyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.Aromatic groups also include heteroaromatic groups such as N-imidazolyl,2-imidazole, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl,3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic, alicyclic, aromatic ring or heteroaryl ring isfused to one or more other heteroaryl or aryl rings. Examples include2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl,2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazole,2-benzooxazole, 2-benzimidazole, 2-quinolinyl, 3-quinolinyl,1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl and 3-isoindolyl.

Suitable substituents for alkyl and aryl groups include —OH, halogen(—Br, —Cl, —I and —F), —O(R′), —O—CO—(R′), —CN, —NO₂, —COOH, ═O, —NH₂,—NH(R′), —N(R′)₂, —COO(R′), —CONH₂, —CONH(R′), —CON(R′)₂, —SH, —S(R′),guanidine, alkyl, and aryl. Each R′ is, independently, an alkyl group oran aromatic group. A substituted alkyl or aryl group can have more thanone substituent.

Also included in the present invention are salts of the disclosedcarboxylic acids. For example, amino acids can also be present in theanionic, or conjugate base, form, in combination with a cation. Suitablecations include alkali metal ions, such as sodium and potassium ions;alkaline earth ions, such as calcium and magnesium ions; andunsubstituted and substituted (primary, secondary, tertiary andquaternary) ammonium ions. Suitable cations also include transitionmetal ions such as manganese, copper, nickel, iron, cobalt, and zinc.Basic groups such as amines can also be protonated with a counter anion,such as hydroxide, halogens (chloride, bromide, and iodide), acetate,formate, citrate, ascorbate, sulfate or phosphate.

Alternative methods for synthesizing 2-alkylcysteines are described inco-pending U.S. patent application Ser. No. 60/381,021, filed May 15,2002, the entire contents of which are incorporated herein by reference.

EXEMPLIFICATION EXAMPLE 1

All compounds were used without further purification. The surfactantsRhodafac RE 610 and Soprophor FL were obtained from Rhône-Poulenc,Surfynol 465 from Air Products, Synperonic NP 10 from ICI and sodiumlauryl sulfate from Fluka. For agitation a shaking machine was used(Buhler KL Tuttlingen). Purities of the resulting crystals were measuredby using a PolarMonitor polarimeter (IBZ Hannover). Ethanol was used asthe solvent. The total crystal quantity was dissolved in a 1 mL cell at20° C.)

45 mg of (R,R)- and (S,S)—amino acid derivatives were dissolved in 1 mlof a mixture of 20% v/v 2-hexanol, 12% v/v Rhodafac RE 610, 6% v/vSoprophor FL and 62% v/v water by heating to 80° C. in a 5 mL vial.After the organic derivative was completely dissolved the microemulsionwas cooled down to room temperature and agitated using a shaking machine(420 rpm). During two hours no spontaneous crystallization was observed.The mixture was then seeded with two drops of a dilute, finely groundsuspension of pure (S,S)—(−) amino acid or its ester crystals grownunder similar conditions. After 2 hours of agitation the resultingcrystals were filtered off, washed with water and dried in a gentlenitrogen stream.

EXAMPLE 2

35 mg of R- andS-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4-carboxylic acidwere dissolved in 1 ml of a mixture of 9% N-methyl-pyrrolidone, 9% v/v2-hexanol, 10% v/v Rhodafac RE 610, 5% v/v Soprophor FL and 68% v/vwater by heating to 50° C. in a 5 mL vial. After the product wascompletely dissolved, the microemulsion was cooled down to roomtemperature and agitated with a shaking machine (350 rpm). During twohours, no spontaneous crystallisation was observed. The mixture was thenseeded with two drops of a dilute, finely ground suspension of pureS-product crystals grown under similar conditions. After two hours ofshaking, the resulting crystals were filtered off, washed with water anddried in a gentle nitrogen stream. The procedure yielded 5.4 mg (15.4%)of colorless crystals, with a greater than 90% purity of the Senantiomer.

EXAMPLE 3

4.00 g (S)-2-methylcysteine hydrochloride (23.3 mmol,1.0 meq) and 3.14 g2,4-dihydroxy benzonitrile (23.3 mmol, 1.0 meq) were suspended in 40 mLethanol. After degassing this mixture with nitrogen (30 min) 4.95 gtriethylamine (6.8 mL, 48.9 mmol, 2.05 meq) were added. The obtainedsuspension was heated under reflux in an atmosphere of nitrogen for 20hours and then cooled to room temperature. From this suspension ethanolwas evaporated under reduced pressure until an oil (20% of the initialvolume) was obtained. This oil was dissolved in 50 mL water. Thesolution was adjusted to pH 7.5 with 1.20 ml 20% KOH and was extractedtwo times each with 20 mL methyl t-butyl ether (MTBE). The aqueous layerwas separated, adjusted with 20% KOH to pH 11 and again extracted twotimes each with 20 mL MTBE. After separating the aqueous layer the pHwas set with concentrated HCl to 7.5 and traces of MTBE were distilledoff. Then the aqueous solution was acidified with 1.50 ml concentratedHCl to pH 1.5. The product precipitated. This suspension was stirred at4° C. for 1 hour. Then the precipitate was filtered, washed two timeseach with 10 mL water (5° C.) and dried at 45° C. under vacuum. Thereaction yielded 5.17 g (87.6%) of crude4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid product. ¹H-NMR showed no significant impurity.

EXAMPLE 4

A single-neck 500 mL round-bottomed flask was flushed with nitrogen.(R)-(+)-L-cysteine hydrochloride monohydrate (12.0 g, 68.32 mmol) wastransferred to the flask. Ethanol (240 mL) was added to give asuspension. Anhydrous triethylamine (34.6 g, 47.7 mL, 341.6 mmol, 5.0equiv.) was then added via a syringe over a period of 10 min. at roomtemperature. A white precipitate of triethylamine hydrochloride formedimmediately. After stirring this thick white turbid solution for 30 min.at room temperature, benzonitrile (7.05 g, 68.32 mmol) was added and thereaction mixture was refluxed for 6 hours. TLC (CH₂Cl₂ as eluent)indicated that all benzonitrile was consumed. The reaction mixture wascooled to room temperature and the solvent was removed in vacuo. Water(25 mL) was added followed by the addition of solid KOH (5 g) withstirring. This reddish clear aqueous solution (pH˜11-12) was extractedwith ethyl acetate (3×100 mL) and the organic layer was discarded. Theaqueous layer was acidified with dropwise addition of 6M HCl to pH1.5-2.0 to obtain an off-white to tan colored precipitate. This solidwas filtered through a Buchner funnel. After drying under high vacuum,the solid was triturated with ethyl acetate to remove any traces ofcolored impurities. After filtration and drying, the off-white to whitesolid was stirred over dichloromethane to remove any traces oftriethylamine hydrochloride and then filtered. After drying undervacuum, a white powdery solid was obtained (10.49 g, 74%).

EXAMPLE 5

2,4-Dibenzyloxybenzonitrile (0.121 mol) was dissolved in 5.85 g (0.127mol) ethanol and 19.4 ml 1,2-dimethoxyethane in a double walled reactor.This solution was cooled to −5° C., stirred and saturated with dry HClgas over 5 hours at 0-3° C. The reaction mixture was stirred overnightat 2-4° C. under nitrogen. During this time, a product crystallized. Thewhite crystals were filtered off, washed with 1,2-dimethoxyethane (5°C., three times each with 13 ml) and dried. A total of 30 of theprotected ethyl benzimidate was isolated (Yield 88.4%, purity 98.9%).

The protected ethyl benzimidate described above was dissolved inmethanol to generate a 10% solution and was catalytically hydrogenatedat room temperature using 5% Pd/C as a catalyst. The reaction wascompleted after 8 hours. The solution was filtered and the solventevaporated to yield the deprotected product as an orange-yellow solid.The reaction yielded 19.6 g (94%) of product.

In contrast, the formation of the imidate with 2,4 dihydroxybenzonitrilewas a low yielding process, generating the desired product in only 20%yield and with less than desired purity.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

We claim:
 1. A method of preparing an optically active 2-alkylatedcysteine represented by Structural Formula (I):

or a salt thereof; wherein R₁ is a substituted or unsubstituted alkylgroup, the method comprising: (a) coupling a compound represented byStructural Formula (II):

 or a salt thereof, with a substituted or unsubstituted aryl nitrile ofthe formula Ar—CN, wherein Ar is a substituted or unsubstituted arylgroup; thereby forming a substituted thiazoline carboxylic acidrepresented by Structural Formula (III):

(b) reacting the substituted thiazoline carboxylic acid with an aminerepresented by Structual Formula (IV):

 wherein R^(*) is a substituted or unsubstituted alkyl group comprisingone or more chiral carbon atoms and R₂ is a substituted or unsubstitutedalkyl or aryl group; thereby forming a substituted thiazoline amiderepresented by Structural Formula (V):

(c) alkylating the substituted thiazoline amide with one or more basesand R₁X, wherein X is a leaving group and R₁ is as defined above;thereby forming an alkylated substituted thiazoline amide represented byStructural Formula (VI):

(d) hydrolyzing the alkylated substituted thiazoline amide, therebyforming an alkylated substituted thiazoline carboxylic acid or a saltthereof, the anion of which is represented by Structural Formula (VII):

(e) reacting the alkylated substituted thiazoline carboxylic acid orsalt thereof with acid, thereby forming the 2-alkylated cysteinerepresented by Structural Formula (I).
 2. The method of claim 1, whereinAr is a substituted or unsubstituted phenyl group.
 3. The method ofclaim 2, wherein Ar is phenyl.
 4. The method of claim 1, wherein theamine represented by Structual Formula (IV) is a compound selected fromthe group consisting of phenylethylamines and heteroaryl chiral amines.5. The method of claim 1, wherein the one or more bases of step (c) areselected from the group consisting of potassium t-butoxide, sodiummethoxide, sodium ethoxide, and sodium amide.
 6. The method of claim 1,wherein R₁ is a substituted or unsubstituted C1-C4 alkyl group.
 7. Themethod of claim 6, wherein R₁ is methyl.
 8. The method of claim 1,wherein X is iodine.
 9. The method of claim 1 further comprising thestep of purifying the alkylated substituted thiazoline amide byresolving the enantiomers of the alkylated substituted thiazoline amide.10. The method of claim 9, wherein the (S)-isomer, based on carbon atom4 as indicated in Structual Formula (VI), of the alkylated substitutedthiazoline amide is isolated.
 11. The method of claim 1 furthercomprising the step of purifying the alkylated substituted thiazolinecarboxylic acid by resolving the enantiomers of the alkylatedsubstituted thiazoline carboxylic acid.
 12. The method of claim 11,wherein the (S)-isomer of the alkylated substituted thiazolinecarboxylic acid is isolated.
 13. A method of preparing a compoundrepresented by Structural Formula (VIII):

or a salt thereof, the method comprising: (a) coupling a compoundrepresented by Structural Formula (IX):

 or a salt thereof, with a substituted or unsubstituted aryl nitrile ofthe formula Ar—CN, wherein Ar is a substituted or unsubstituted arylgroup; thereby forming a substituted thiazoline carboxylic acidrepresented by Structural Formula (X):

(b) reacting the substituted thiazoline carboxylic acid with an aminerepresented by Structual Formula (XI):

 wherein R^(*) is a substituted or unsubstituted alkyl group comprisingone or more chiral carbon atoms and R₂ is a substituted or unsubstitutedalkyl or aryl group; thereby forming a substituted thiazoline amiderepresented by Structural Formula (XII):

(c) alkylating the substituted thiazoline amide with one or more basesand CH₃X, wherein X is a leaving group;  thereby forming an alkylatedsubstituted thiazoline amide represented by Structural Formula (XIII):

(d) hydrolyzing the alkylated substituted thiazoline amide, therebyforming an alkylated substituted thiazoline carboxylic acid or a saltthereof, the anion of which is represented by Structural Formula (XIV):

(e) optionally, purifying the (S)-isomer of the alkylated substitutedthiazoline carboxylic acid; (f) reacting the (S)-isomer of the alkylatedsubstituted thiazoline carboxylic acid with acid, thereby forming(S)-2-methylcysteine; and (g) coupling (S)-2-methylcysteine with2,4-dihydroxybenzonitrile, thereby forming the compound represented byStructural Formula (VIII).